基于酶促重组等温扩增与 CRISPR/ EsCas13d 技术构建的猪圆环病毒 4型快速可视化检测方法

A rapid visual detection method for porcine circovirus type 4 based on enzymatic recombinase amplification and CRISPR/ EsCas13d

摘要:

人类与动物传染病在全球范围内持续暴发，严重威胁公共卫生安全并影响经济发展，因此亟需更加高效、灵敏的诊断方法来提升疫情监测与预警能力。本研究旨在开发一种基于ERA-CRISPR/ EsCas13d的便携式可视化平台，于资源有限的环境中实现对猪圆环病毒4型的快速检测。对该平台进行了体系优化，使酶促重组等温扩增技术(enzymatic recombinase amplification, ERA)、T7转录以及CRISPR/ EsCas13d切割反应能够在单管内连续完成，极大简化了操作流程，并将总检测时间缩短至30 min。同时，配套的快速核酸释放方法无需实验室提取及复杂的加热步骤，进一步提升了平台的简便性与实用性。采用改良的冻干试剂，降低了冷链运输和储存需求，简化了制备流程，便于实现低成本运输与现场部署。该方法可灵敏检测猪圆环病毒4型，可视化检测限为50 cp/μL；在特异性评价中，对其他6种常见猪病原均未检测到信号(均为阴性)。此外，本平台支持紫外光(470 nm)和侧向层析试纸条(lateral flow assay, LFA) 2种可视化读数方式，能够在不同应用场景下灵活判读。通过对60份临床样本的验证，检测结果与荧光定量PCR符合率为100%，充分展示了ERA-CRISPR/ EsCas13d便携式可视化平台在现场快速检测与便携式诊断中的应用前景与实用价值。

Abstract:

The emerging outbreaks of infectious diseases in humans and animals worldwide pose serious threats to public health and cause substantial economic losses. This has raised the demand for more efficient and sensitive diagnostic methods to strengthen disease surveillance and early warning. In this study, we developed a portable visual platform based on ERA-CRISPR/ EsCas13d for the rapid detection of porcine circovirus type 4 under resource-limited conditions. We optimized the platform by integrating enzymatic recombinase amplification (ERA), T7 transcription, and CRISPR/ EsCas13d cleavage in a single-tube reaction, thereby simplifying the workflow and shortening the total detection time to 30 min. In addition, a rapid nucleic acid release method was employed, eliminating the need for laboratory-based extraction and complex heating steps, which further improved the simplicity and usability. In addition, lyophilized reagents were modified to enhance stability, thus reducing cold-chain and storage requirements and enabling cost-effective transport and field deployment. The method achieved a visual limit of detection of 50 cp/μL for porcine circovirus type 4 and demonstrated no cross-reactivity with six other common swine pathogens. Moreover, the platform supported two visual readout formats—UV light (470 nm) and lateral flow assay (LFA)—providing flexible options for interpretation in different application scenarios. Validation with 60 clinical samples showed 100% concordance with quantitative PCR results, highlighting the practical utility and application potential of the ERA-CRISPR/ EsCas13d-based portable visual platform for rapid on-site diagnostics.